Zyxin is not colocalized with vasodilator-stimulated phosphoprotein (VASP)at lamellipodial tips and exhibits different dynamics to vinculin, paxillin, and VASP in focal adhesions

Actin polymerization is accompanied by the formation of protein complexes t hat link extracellular signals to sites of actin assembly such as membrane ruffles and focal adhesions. One candidate recently implicated in these pro cesses is the LIM domain protein zyxin, which can bind both Ena/vasodilator -stimulated phosphoprotein (VASP) proteins and the actin filament cross-lin king protein a-actinin. To characterize the localization and dynamics of zy xin in detail, we generated both monoclonal antibodies and a green fluoresc ent protein (GFP)-fusion construct. The antibodies colocalized with ectopic ally expressed GFP-VASP at focal adhesions and along stress fibers, but fai led to label lamellipodial and filopodial tips, which also recruit Ena/VASP proteins. Likewise, neither microinjected, fluorescently labeled zyxin ant ibodies nor ectopically expressed GFP-zyxin were recruited to these latter sites in live cells, whereas both probes incorporated into focal adhesions and stress fibers. Comparing the dynamics of zyxin with that of the focal a dhesion protein vinculin revealed that both proteins incorporated simultane ously into newly formed adhesions. However, during spontaneous or induced f ocal adhesion disassembly, zyxin delocalization preceded that of either vin culin or paxiliin. Together, these data identify zyxin as an early target f or signals leading to adhesion disassembly, but exclude its role in recruit ing Ena/VASP proteins to the tips of lamellipodia and filopodia.